Type I interferons and the cytokine TNF cooperatively reprogram the macrophage epigenome to promote inflammatory activation.

Cross-regulation of Toll-like receptor (TLR) responses by cytokines is essential for effective host defense, avoidance of toxicity and homeostasis, but the underlying mechanisms are not well understood. Our comprehensive epigenomics approach to the analysis of human macrophages showed that the proinflammatory cytokines TNF and type I interferons induced transcriptional cascades that altered chromatin states to broadly reprogram responses induced by TLR4. TNF tolerized genes encoding inflammatory molecules to prevent toxicity while preserving the induction of genes encoding antiviral and metabolic molecules. Type I interferons potentiated the inflammatory function of TNF by priming chromatin to prevent the silencing of target genes of the transcription factor NF-κB that encode inflammatory molecules. The priming of chromatin enabled robust transcriptional responses to weak upstream signals. Similar chromatin regulation occurred in human diseases. Our findings reveal that signaling crosstalk between interferons and TNF is integrated at the level of chromatin to reprogram inflammatory responses, and identify previously unknown functions and mechanisms of action of these cytokines.

The Cytokine TNF Promotes Transcription Factor SREBP Activity and Binding to Inflammatory Genes to Activate Macrophages and Limit Tissue Repair.

Cytokine tumor necrosis factor (TNF)-mediated macrophage polarization is important for inflammatory disease pathogenesis, but the mechanisms regulating polarization are not clear. We performed transcriptomic and epigenomic analysis of the TNF response in primary human macrophages and revealed late-phase activation of SREBP2, the master regulator of cholesterol biosynthesis genes. TNF stimulation extended the genomic profile of SREBP2 occupancy to include binding to and activation of inflammatory and interferon response genes independently of its functions in sterol metabolism. Genetic ablation of SREBP function shifted the balance of macrophage polarization from an inflammatory to a reparative phenotype in peritonitis and skin wound healing models. Genetic ablation of SREBP activity in myeloid cells or topical pharmacological inhibition of SREBP improved skin wound healing under homeostatic and chronic inflammatory conditions. Our results identify a function and mechanism of action for SREBPs in augmenting TNF-induced macrophage activation and inflammation and open therapeutic avenues for promoting wound repair.

Interferon-γ Represses M2 Gene Expression in Human Macrophages by Disassembling Enhancers Bound by the Transcription Factor MAF.

Mechanisms by which interferon (IFN)-γ activates genes to promote macrophage activation are well studied, but little is known about mechanisms and functions of IFN-γ-mediated gene repression. We used an integrated transcriptomic and epigenomic approach to analyze chromatin accessibility, histone modifications, transcription-factor binding, and gene expression in IFN-γ-primed human macrophages. IFN-γ suppressed basal expression of genes corresponding to an "M2"-like homeostatic and reparative phenotype. IFN-γ repressed genes by suppressing the function of enhancers enriched for binding by transcription factor MAF. Mechanistically, IFN-γ disassembled a subset of enhancers by inducing coordinate suppression of binding by MAF, lineage-determining transcription factors, and chromatin accessibility. Genes associated with MAF-binding enhancers were suppressed in macrophages isolated from rheumatoid-arthritis patients, revealing a disease-associated signature of IFN-γ-mediated repression. These results identify enhancer inactivation and disassembly as a mechanism of IFN-γ-mediated gene repression and reveal that MAF regulates the macrophage enhancer landscape and is suppressed by IFN-γ to augment macrophage activation.

Hypoxia-Sensitive COMMD1 Integrates Signaling and Cellular Metabolism in Human Macrophages and Suppresses Osteoclastogenesis.

Hypoxia augments inflammatory responses and osteoclastogenesis by incompletely understood mechanisms. We identified COMMD1 as a cell-intrinsic negative regulator of osteoclastogenesis that is suppressed by hypoxia. In human macrophages, COMMD1 restrained induction of NF-κB signaling and a transcription factor E2F1-dependent metabolic pathway by the cytokine RANKL. Downregulation of COMMD1 protein expression by hypoxia augmented RANKL-induced expression of inflammatory and E2F1 target genes and downstream osteoclastogenesis. E2F1 targets included glycolysis and metabolic genes including CKB that enabled cells to meet metabolic demands in challenging environments, as well as inflammatory cytokine-driven target genes. Expression quantitative trait locus analysis linked increased COMMD1 expression with decreased bone erosion in rheumatoid arthritis. Myeloid deletion of Commd1 resulted in increased osteoclastogenesis in arthritis and inflammatory osteolysis models. These results identify COMMD1 and an E2F-metabolic pathway as key regulators of osteoclastogenic responses under pathological inflammatory conditions and provide a mechanism by which hypoxia augments inflammation and bone destruction.

Inhibition of sodium-glucose cotransporter-2 and liver-related complications in individuals with diabetes: a Mendelian randomization and population-based cohort study.

BACKGROUND AND AIMS: No medication has been found to reduce liver-related events. We evaluated the effect of sodium-glucose cotransporter-2 inhibitor (SGLT2i) on liver-related outcomes. APPROACH AND RESULTS: Single nucleotide polymorphisms associated with SGLT2 inhibition were identified, and a genetic risk score (GRS) was computed using the UK Biobank data (n=337,138). Two-sample Mendelian randomization (MR) was conducted using the FinnGen (n=218,792) database and the UK Biobank data. In parallel, a nationwide population-based study using the Korean National Health Insurance Service (NHIS) database was conducted. The development of liver-related complications (ie, hepatic decompensation, HCC, liver transplantation, and death) was compared between individuals with type 2 diabetes mellitus and steatotic liver diseases treated with SGLT2i (n=13,208) and propensity score-matched individuals treated with dipeptidyl peptidase-4 inhibitor (n=70,342). After computing GRS with 6 single nucleotide polymorphisms (rs4488457, rs80577326, rs11865835, rs9930811, rs34497199, and rs35445454), GRS-based MR showed that SGLT2 inhibition (per 1 SD increase of GRS, 0.1% lowering of HbA1c) was negatively associated with cirrhosis development (adjusted odds ratio=0.83, 95% CI=0.70-0.98, p =0.03) and this was consistent in the 2-sample MR (OR=0.73, 95% CI=0.60-0.90, p =0.003). In the Korean NHIS database, the risk of liver-related complications was significantly lower in the SGLT2i group than in the dipeptidyl peptidase-4 inhibitor group (adjusted hazard ratio=0.88, 95% CI=0.79-0.97, p =0.01), and this difference remained significant (adjusted hazard ratio=0.72-0.89, all p <0.05) across various sensitivity analyses. CONCLUSIONS: Both MRs using 2 European cohorts and a Korean nationwide population-based cohort study suggest that SGLT2 inhibition is associated with a lower risk of liver-related events.

Metabolic effects and cardiovascular disease risks of antiviral treatments in patients with chronic hepatitis B.

Different antiviral treatments for chronic hepatitis B (CHB) have been known to have different metabolic effects. This study aimed to reveal whether tenofovir alafenamide (TAF)-induced dyslipidemia and its associated outcomes are significant. This study utilized 15-year historical cohort including patients with CHB in Korea and consisted of two parts: the single-antiviral and switch-antiviral cohorts. In the single-antiviral cohort, patients were divided into four groups (entecavir [ETV]-only, tenofovir disoproxil fumarate [TDF]-only, TAF-only, and non-antiviral). Propensity score matching (PSM) and linear regression model were sequentially applied to compare metabolic profiles and estimated atherosclerotic cardiovascular disease (ASCVD) risks longitudinally. In the switch-antiviral cohort, pairwise analyses were conducted in patients who switched NAs to TAF or from TAF. In the single-antiviral cohort, body weight and statin use showed significant differences between groups before PSM, but well-balanced after PSM. Changes in total cholesterol were significantly different between groups (-2.57 mg/dL/year in the TDF-only group and +2.88 mg/dL/year in the TAF-only group; p = 0.002 and p = 0.02, respectively). In the TDF-only group, HDL cholesterol decreased as well (-0.55 mg/dL/year; p < 0.001). The TAF-only group had the greatest increase in ASCVD risk, followed by the TDF-only group and the non-antiviral group. In the switch-antiviral cohort, patients who switched from TDF to TAF had a higher total cholesterol after switching (+9.4 mg/dL/year) than before switching (-1.0 mg/dL/year; p = 0.047). Sensitivity analysis on data with an observation period set to a maximum of 3 years for NA treatment showed consistent results on total cholesterol (-2.96 mg/dL/year in the TDF-only group and +3.09 mg/dL/year in the TAF-only group; p = 0.001 and p = 0.005, respectively). Another sensitivity analysis conducted on statin-treated patients revealed no significant change in cholesterol and ASCVD risk. TAF was associated with increased total cholesterol, whereas TDF was associated with decreased total and HDL cholesterol. Both TAF and TDF were associated with increased ASCVD risks, and statin use might mitigate these risks.

Extrahepatic malignancies in metabolic dysfunction-associated fatty liver disease: A nationwide cohort study.

BACKGROUND AND AIMS: Metabolic dysfunction-associated fatty liver disease (MAFLD) encompasses heterogeneous fatty liver diseases associated with metabolic disorders. We aimed to evaluate the association between MAFLD and extrahepatic malignancies based on MAFLD subtypes. METHODS: This nationwide cohort study included 9 298 497 patients who participated in a health-screening programme of the National Health Insurance Service of Korea in 2009. Patients were further classified into four subgroups: non-MAFLD, diabetes mellitus (DM)-MAFLD, overweight/obese-MAFLD and lean-MAFLD. The primary outcome was the development of any primary extrahepatic malignancy, while death, decompensated liver cirrhosis and liver transplantation were considered competing events. The secondary outcomes included all-cause and extrahepatic malignancy-related mortality. RESULTS: In total, 2 500 080 patients were diagnosed with MAFLD. During a median follow-up of 10.3 years, 447 880 patients (6.0%) with extrahepatic malignancies were identified. The DM-MAFLD (adjusted subdistribution hazard ratio [aSHR] = 1.13; 95% confidence interval [CI] = 1.11-1.14; p < .001) and the lean-MAFLD (aSHR = 1.12; 95% CI = 1.10-1.14; p < .001) groups were associated with higher risks of extrahepatic malignancy than the non-MAFLD group. However, the overweight/obese-MAFLD group exhibited a similar risk of extrahepatic malignancy compared to the non-MAFLD group (aSHR = 1.00; 95% CI = .99-1.00; p = .42). These findings were reproduced in several sensitivity analyses. The DM-MAFLD was an independent risk factor for all-cause mortality (adjusted hazard ratio [aHR] = 1.41; 95% CI = 1.40-1.43; p < .001) and extrahepatic malignancy-related mortality (aHR = 1.20; 95% CI = 1.17-1.23; p < .001). CONCLUSION: The diabetic or lean subtype of MAFLD was associated with a higher risk of extrahepatic malignancy than non-MAFLD. As MAFLD comprises a heterogeneous population, appropriate risk stratification and management based on the MAFLD subtypes are required.

WNT-modulating gene silencers as a gene therapy for osteoporosis, bone fracture, and critical-sized bone defects.

Treating osteoporosis and associated bone fractures remains challenging for drug development in part due to potential off-target side effects and the requirement for long-term treatment. Here, we identify recombinant adeno-associated virus (rAAV)-mediated gene therapy as a complementary approach to existing osteoporosis therapies, offering long-lasting targeting of multiple targets and/or previously undruggable intracellular non-enzymatic targets. Treatment with a bone-targeted rAAV carrying artificial microRNAs (miRNAs) silenced the expression of WNT antagonists, schnurri-3 (SHN3), and sclerostin (SOST), and enhanced WNT/ß-catenin signaling, osteoblast function, and bone formation. A single systemic administration of rAAVs effectively reversed bone loss in both postmenopausal and senile osteoporosis. Moreover, the healing of bone fracture and critical-sized bone defects was also markedly improved by systemic injection or transplantation of AAV-bound allograft bone to the osteotomy sites. Collectively, our data demonstrate the clinical potential of bone-specific gene silencers to treat skeletal disorders of low bone mass and impaired fracture repair.

Personalized Antiviral Drug Selection in Patients With Chronic Hepatitis B Using a Machine Learning Model: A Multinational Study.

INTRODUCTION: Tenofovir disoproxil fumarate (TDF) is reportedly superior or at least comparable to entecavir (ETV) for the prevention of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B; however, it has distinct long-term renal and bone toxicities. This study aimed to develop and validate a machine learning model (designated as Prediction of Liver cancer using Artificial intelligence-driven model for Network-antiviral Selection for hepatitis B [PLAN-S]) to predict an individualized risk of HCC during ETV or TDF therapy. METHODS: This multinational study included 13,970 patients with chronic hepatitis B. The derivation (n = 6,790), Korean validation (n = 4,543), and Hong Kong-Taiwan validation cohorts (n = 2,637) were established. Patients were classified as the TDF-superior group when a PLAN-S-predicted HCC risk under ETV treatment is greater than under TDF treatment, and the others were defined as the TDF-nonsuperior group. RESULTS: The PLAN-S model was derived using 8 variables and generated a c-index between 0.67 and 0.78 for each cohort. The TDF-superior group included a higher proportion of male patients and patients with cirrhosis than the TDF-nonsuperior group. In the derivation, Korean validation, and Hong Kong-Taiwan validation cohorts, 65.3%, 63.5%, and 76.4% of patients were classified as the TDF-superior group, respectively. In the TDF-superior group of each cohort, TDF was associated with a significantly lower risk of HCC than ETV (hazard ratio = 0.60-0.73, all P < 0.05). In the TDF-nonsuperior group, however, there was no significant difference between the 2 drugs (hazard ratio = 1.16-1.29, all P > 0.1). DISCUSSION: Considering the individual HCC risk predicted by PLAN-S and the potential TDF-related toxicities, TDF and ETV treatment may be recommended for the TDF-superior and TDF-nonsuperior groups, respectively.

Effectiveness of US Surveillance of Hepatocellular Carcinoma in Chronic Hepatitis B: US LI-RADS Visualization Score.

Background US is a standard surveillance tool of hepatocellular carcinoma (HCC), but its effectiveness varies depending on the degree of fibrosis or steatosis and the etiologies of liver disease. Purpose To evaluate the detection power of US and the occurrence of HCC according to the US Liver Imaging Reporting and Data System (LI-RADS) visualization score in chronic hepatitis B (CHB). Materials and Methods Consecutive patients with CHB undergoing regular US surveillance of HCC at a tertiary referral hospital were retrospectively included in this study. During the follow-up, all patients underwent regular HCC surveillance mainly with US and, in some cases, alternative CT or MRI. Outcomes of interest included cumulative incidence of HCC and false-negative rate of US in the optimal (LI-RADS visualization A) versus suboptimal groups (visualization B or C). Cox regression analysis was conducted to calculate the hazard ratio (HR) of HCC occurrence. Results A total of 2002 patients (median age, 54 years [IQR, 46-60 years]; 1192 men) were included: 972 and 1030 in the optimal and suboptimal groups, respectively. Causes of suboptimal visualization included parenchymal heterogeneity from advanced cirrhosis (n = 489), limited penetration from fatty liver (n = 200), and limited window from overlying organ shadow (n = 341). During a median follow-up of 75 months (IQR, 69-77 months), 163 patients developed HCC. Compared with the optimal group, the suboptimal group had a higher risk of HCC (2.38% per year vs 0.48% per year: hazard ratio, 4.93; 95% CI: 3.28, 7.41; P < .001) and higher odds of a false-negative rate of US (43.9% vs 16.7%: odds ratio, 3.90; 95% CI: 1.02, 15.00; P = .04). Conclusion Among patients with CHB, those with suboptimal US LI-RADS visualization of B or C had a higher risk of HCC and higher odds of false-negative rates of US for detecting HCC than those with optimal visualization of A. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Barr and Scoutt in this issue.

Tumor necrosis factor induces GSK3 kinase-mediated cross-tolerance to endotoxin in macrophages.

Endotoxin tolerance, a key mechanism for suppressing excessive inflammatory cytokine production, is induced by prior exposure of macrophages to Toll-like receptor (TLR) ligands. Induction of cross-tolerance to endotoxin by endogenous cytokines has not been investigated. Here we show that prior exposure to tumor necrosis factor (TNF) induced a tolerant state in macrophages, with less cytokine production after challenge with lipopolysaccharide (LPS) and protection from LPS-induced death. TNF-induced cross-tolerization was mediated by suppression of LPS-induced signaling and chromatin remodeling. TNF-induced cross-tolerance was dependent on the kinase GSK3, which suppressed chromatin accessibility and promoted rapid termination of signaling via the transcription factor NF-κB by augmenting negative feedback by the signaling inhibitors A20 and IκBα. Our results demonstrate an unexpected homeostatic function for TNF and a GSK3-mediated mechanism for the prevention of prolonged and excessive inflammation.

The association between depression and non-compliance with COVID-19 preventive behaviors in South Korean older adults stratified by sex.

OBJECTIVES: This study evaluated the association between depression and non-compliance with COVID-19 preventive behaviors among community-dwelling South Korean older adults. METHODS: We utilized the 2020 Korean Community Health Survey-a community-based nationwide survey. A score of 10 points or higher on the Patient Health Questionnaire-9 was defined as depression. Non-compliance with COVID-19 preventive behaviors was assessed on the following three behaviors: washing hands, wearing masks, and watching distance. We also included socio-demographic characteristics, health behaviors, and COVID-19-related characteristics as covariates. Multiple logistic regression analyses were performed, and all statistical analyses were stratified by sex. RESULTS: The 70,693 participants included 29,736 men and 40,957 women. Notably, 2.3% of men and 4.2% of women had depression. Non-compliance with washing hands was significantly higher in men than women (1.3% vs. 0.9%), whereas no significant differences were observed in wearing masks and watching distance. The adjusted logistic regression analysis showed that depression was positively associated with non-compliance with washing hands and watching distance in both sexes. The association between depression and non-compliance with wearing masks was significant only in women. CONCLUSIONS: There was an association between depression and non-compliance with COVID-19 preventive behaviors in South Korean older adults. This signifies that health providers need to reduce depression to improve compliance with preventive behaviors in older adults.

Two-year longitudinal associations between nutritional status and frailty in community-dwelling older adults: Korean Frailty and Aging Cohort Study.

BACKGROUND: Korea is expected to become a super-aged society in 2026, and improving nutritional status, which is directly related to health problems, is therefore important for increasing healthy life expectancy. Frailty is the most complex phenotype of aging, and leads to adverse health outcomes, disability, poor quality of life, hospitalization, and mortality. Malnutrition is a major risk factor for frailty syndrome. This study aimed to investigate the incidence of pre-frailty or frailty in the second wave (T2, 2018-2019) according to general characteristics and nutritional status in the first wave (T1, 2016-2017); and examine the longitudinal association of nutritional status in T1 and the incidence of pre-frailty or frailty in T2 among older adults living in a community. METHODS: A secondary data analysis was performed using the Korean Frailty and Aging Cohort Study (KFACS). Participants comprised 1125 community-dwelling older Korean adults aged 70-84 years (mean age: 75.03 ± 3.56 years; 53.8% males). Frailty was assessed using the Fried frailty index, and nutritional status was assessed using the Korean version of the Mini Nutritional Assessment Short-Form and blood nutritional biomarkers. Binary logistic regression was used to identify longitudinal associations between the nutritional status at T1 and pre-frailty or frailty at T2. RESULTS: Over the two-year follow-up period, 32.9% and 1.7% of the participants became pre-frail and frail, respectively. After the potential confounders were adjusted (sociodemographic, health behaviors, and health status characteristics), pre-frailty or frailty had a significant longitudinal association with severe anorexia (adjusted odds ratio [AOR], 4.17; 95% confidence interval [CI], 1.05-16.54), moderate anorexia (AOR, 2.31; 95% CI, 1.46-3.64), psychological stress or acute disease (AOR, 2.61; 95% CI, 1.26-5.39), and body mass index (BMI) less than 19 (AOR, 4.11; 95% CI, 1.20-14.04). CONCLUSIONS: Anorexia, psychological stress, acute disease, and low BMI are the most significant longitudinal risk factors for pre-frailty or frailty in older adults. As nutritional risk factors may be preventable or modifiable, it is important to develop interventions targeting the same. Community-based health professionals in health-related fields should recognize and manage these indicators appropriately to prevent frailty among older adults living in the community.

Comparison of the outcomes between sorafenib and lenvatinib as the first-line systemic treatment for HBV-associated hepatocellular carcinoma: a propensity score matching analysis.

BACKGROUND/AIM: In a randomized controlled trial, lenvatinib was non-inferior to sorafenib in overall survival (OS) of patients with unresectable hepatocellular carcinoma (uHCC). This study aimed to compare the effects of sorafenib and lenvatinib as first-line systemic therapy against uHCC with real-world data in chronic hepatitis B patients. METHODS: This retrospective single-center study involved 132 patients with HBV-related uHCC. Propensity score matching (PSM) was used to balance the baseline characteristics, including age, sex, serum alpha-fetoprotein levels, Child-Pugh class, tumor size, and tumor stage. The primary endpoint was overall survival (OS), and the secondary endpoints included progression-free survival (PFS), time to progression (TTP), and tumor response. RESULTS: After PSM, the final analysis included 44 patients treated with lenvatinib and 88 with sorafenib. The OS (7.0 vs 9.2 months, p = 0.070) and PFS (4.6 vs 2.4 months, p = 0.134) were comparable between the two drugs. Multivariable analysis showed that lenvatinib and sorafenib were not independent prognostic factors of OS (adjusted hazard ratio = 1.41, 95% confidence interval = 0.96-2.08, p = 0.077) after adjustment for baseline alpha-fetoprotein levels, total bilirubin levels, alanine aminotransferase level, performance status, tumor stage, and tumor size. However, the lenvatinib group had a significantly prolonged TTP (5.2 vs 2.5 months, p = 0.018) and a higher objective response rate (18.2% vs 4.5%, p = 0.020) and disease control rate (77.3% vs 47.7%, p = 0.001) than the sorafenib group. CONCLUSIONS: Our study demonstrated that lenvatinib had a comparable OS and PFS but longer TTP and better tumor response compared to sorafenib in patients with HBV-related uHCC.

Effectiveness of Lenvatinib Versus Sorafenib for Unresectable Hepatocellular Carcinoma in Patients with Hepatic Decompensation.

BACKGROUND/AIM: Lenvatinib and sorafenib are currently available to treat patients with advanced hepatocellular carcinoma (HCC). However, since the clinical trials evaluating the efficacy of lenvatinib and sorafenib included only patients with Child-Pugh class A, little is known about the effectiveness of the treatments in patients with hepatic decompensation. We compared the effectiveness of lenvatinib and sorafenib in decompensated patients with unresectable HCC. METHODS: Consecutive patients who were classified as Child-Pugh class B or C and received lenvatinib or sorafenib as first-line systemic therapy for unresectable HCC between November 2018 and April 2020 at a tertiary referral center were included in this retrospective study. The primary outcome was overall survival (OS), and the secondary outcomes were progression-free survival (PFS), time-to-progression, best overall tumor response, and safety profiles. RESULTS: Among 94 patients, 34 received lenvatinib and 60 received sorafenib. The median OS was 4.1 months (95% confidence interval [CI], 2.9-5.2): 4.2 months (95% CI, 2.9-5.3) for lenvatinib and 4.1 months (95% CI, 2.7-6.4) for sorafenib. The treatment regimen was not associated with significant improvement in OS after adjusting for covariables (adjusted hazard ratio [aHR], 0.92; 95% CI, 0.54-1.54; P = 0.74). The treatment regimen was not an independent predictor of PFS (lenvatinib vs. sorafenib; aHR, 0.77; 95% CI, 0.48-1.24; P = 0.28). HRs were maintained even after balancing with the inverse probability treatment weighting method. Objective response rates were 11.8% and 6.7% in patients receiving lenvatinib and sorafenib, respectively (P = 0.45). Ten patients in both groups (five in the lenvatinib group and five in the sorafenib group) underwent dose modification due to adverse events, and significant difference was not observed between the treatment groups (P = 0.49). CONCLUSION: The effectiveness of lenvatinib and sorafenib was comparable for the treatment of unresectable HCC in decompensated patients.

Cancer-specific cytotoxicity of pyridinium-based ionic liquids by regulating hypoxia-inducible factor-1α-centric cancer metabolism.

Owing to their unique properties and biological activities, ionic liquids (ILs) have attracted research interest in pharmaceutics and medicine. Hypoxia-inducible factor (HIF)- 1α is an attractive cancer drug target involved in cancer malignancy in the hypoxic tumor microenvironment. Herein, we report the inhibitory activity of ILs on the HIF-1α pathway and their mechanism of action. Substitution of a dimethylamino group on pyridinium reduced hypoxia-induced HIF-1α activation. It selectively inhibited the viability of the human colon cancer cell line HCT116, compared to that of the normal fibroblast cell line WI-38. These activities were enhanced by increasing the alkyl chain length in the pyridinium. Under hypoxic conditions, dimethylaminopyridinium reduced the accumulation of HIF-1α and its target genes without affecting the HIF1A mRNA level in cancer cells. It suppressed the oxygen consumption rate and ATP production by directly inhibiting electron transfer chain complex I, which led to enhanced intracellular oxygen content and oxygen-dependent degradation of HIF-1α under hypoxia. These results indicate that dimethylaminopyridinium suppresses the mitochondria and HIF-1α-dependent glucose metabolic pathway in hypoxic cancer cells. This study provides insights into the anticancer activity of pyridinium-based ILs through the regulation of cancer metabolism, making them promising candidates for cancer treatment.

Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture.

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is mainly unknown. Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 × 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 × 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 × 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

TNF activates an IRF1-dependent autocrine loop leading to sustained expression of chemokines and STAT1-dependent type I interferon-response genes.

Rapid induction of inflammatory genes by tumor necrosis factor (TNF) has been well studied, but little is known about delayed and chronic TNF responses. Here we investigated the kinetics of primary macrophage responses to TNF and discovered that TNF initiates an interferon-beta-mediated autocrine loop that sustains expression of inflammatory genes and induces delayed expression of interferon-response genes such as those encoding the transcription factors STAT1 and IRF7, which enhance macrophage responses to stimulation of cytokines and Toll-like receptors. TNF-induced interferon-beta production depended on interferon-response factor 1, and downstream gene expression was mediated by synergy between small amounts of interferon-beta and canonical TNF-induced signals. Thus, TNF activates a 'feed-forward' loop that sustains inflammation but avoids the potential toxicity associated with the high interferon production induced by stimulation of Toll-like receptors.

'Tuning' of type I interferon-induced Jak-STAT1 signaling by calcium-dependent kinases in macrophages.

Immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors modulate the amplitude and nature of macrophage responses to Toll-like receptor and cytokine receptor stimulation. However, the molecular mechanisms enabling this receptor crosstalk are not known. Here we investigated the function of the calcium-dependent kinases CaMK and Pyk2 'downstream' of ITAM-associated receptors in the regulation of cytokine-induced activation of Jak kinases and STAT transcription factors. CaMK and Pyk2 relayed signals from integrins and the ITAM-containing adaptor DAP12 to augment interleukin 10- and interferon-alpha-induced Jak activation and STAT1-dependent gene expression. CaMK inhibition suppressed STAT1-mediated interferon-alpha signaling in a mouse model of systemic lupus erythematosus. Our results associate Pyk2 and Jak kinases with the linkage of signals emanating from cytokine and heterologous ITAM-dependent receptors.